Use of paullone derivatives for making medicines

ABSTRACT

The invention concerns the use for producing of GSK-3β inhibiting medicines from paullone derivatives. The invention is useful for treating pathologies involving GSK-3β and CDK5.

The invention relates to a new use of paullone derivatives intherapeutics.

The paullones constitute a family belonging to the benzazepinones.

The leader of this family, called paullone, corresponds to the followingformula:

In the Journal of Medicinal Chemistry, 1999, Vol. 42, No. 15, pages2909–2919, the authors, one of whom is a co-inventor of the presentapplication, report the inhibitory properties on cyclin-dependentkinases (abbreviated to CDKs) exhibited by paullones, and theirantitumour activity in vitro.

The CDKs play a major role notably in regulation of the cell cycle, bycontrolling transmission between the successive stages of the cellcycle. Their activity is regulated by a great many mechanisms and inparticular by binding to cyclins which vary during the cell cycle.Binding to CDK inhibitors leads to deactivation of the CDKs.

Paullone derivatives substituted in various positions, and especially inposition 9, have proved to be, as reported in the aforementionedarticle, strong inhibitors of CDK1/-cyclin B. Thus,9-nitro-7,12-dihydroindolo[3,2-d] [1]-benzazepin-6 (5H)-one, calledalsterpaullone, has an IC₅₀ of 0.035 μM and an antitumour activity invitro an order of magnitude greater than 1 (mean log IC₅₀ of themid-point of the graph=−6.4M).

These inhibitory properties of CDKs, which lead to arrest of the cellcycle, mean that paullone derivatives are of interest for the treatmentof pathologies connected with loss of control of proliferation, such ascancers, psoriasis, cardiovascular diseases, infectious diseases,nephrology, neurodegenerative diseases and viral infections.

Surprisingly, the inventors have now demonstrated that some of thesepaullone derivatives exerted an inhibitory effect on another enzymatictarget, namely glycogen synthase kinase-3β or GSK-3β for short, as wellas on the CDKs if appropriate, and especially CDK5/p25.

GSK-3β is an essential element of the WNT signal pathway. It is involvedin numerous physiological processes: regulation of the cell cycle bycontrolling levels of cyclin D1 and of β-catenin, dorso-ventralformation during development, action of insulin on glycogen synthesis,axonal excrescence, Tat-mediated HIV-1 neurotoxicity, and others.

Moreover, it is known that GSK-3p and CDK5 are responsible for a goodproportion of abnormal hyperphosphorylation of the microtubule-bindingtau protein, as observed in the paired helical filaments in Alzheimer'sdisease and other neurodegenerative “taupathies”.

The advantage of having derivatives that are inhibitors of GSK-3βactivity for promoting cell division is also being assessed.

The only inhibitors of GSK-3β disclosed to date comprise lithium andcertain derivatives of purine.

The selectivity of lithium has not been reported, but in view of theatomic nature of the product it is probable that it should be very low.Moreover, lithium is only active at considerable doses (IC₅₀ around 10mM).

The same applies to the purine derivatives described in application WO98/16528, which are of low selectivity and have IC₅₀ values around 10μM.

The invention provides a solution to these problems with the use, formanufacturing medicines that are inhibitors of GSK-3β and if appropriateof CDKs, of paullones of high efficacy, having IC₅₀ values with respectto GSK-3β of less than 10 μM, and even below 5 μM for a good number ofthem, and even less than 1 μM. Some of these compounds even have IC₅₀values below 50 nM, even reaching values below 10 nM for some.

In accordance with the invention, for the manufacture of the saidmedicines with inhibitory effect, notably of GSK-3β, paullonederivatives are used that correspond to general formula (I):

in which

-   -   X represents a C═O, C—S—CH₃, C—S, or —C—NHOH group;    -   Z represents C or N;    -   Y represents, with the adjacent ring, a phenyl or thiazolyl        residue;

the ring or rings constituting these derivatives being substituted ifappropriate by one or more: halogen atoms, hydroxy, alkylenehydroxy,alkynealkylenehydroxy, alkynehydroxycyclohexyl, alkyl, alkoxy,alkylenealkoxy, alkylenecyano groups, the alkylene group being saturatedor unsaturated, the said radicals being straight-chain or branched, ofC1 to C18, the said chain being substituted if appropriate by one ormore hydroxy or amino groups; one or more trifluoromethyl; —COM; —COOM;or —CH₂COOM groups (with M representing a hydrogen atom, a C1 to C18alkyl group, straight-chain or branched, substituted if necessary by oneor more hydroxy and/or amino; nitroso; nitro; or cyano groups;

-   -   R⁵ represents a hydrogen atom or a C₁ to C₅ alkyl group,    -   R¹² represents a hydrogen atom, or a —C—CO₂—(CH₃)₃ group,

and the physiologically acceptable salts of these derivatives.

In a preferred family, Y represents, with the adjacent ring, a phenylresidue, and Z═C. This family corresponds to general formula (II):

in which,

-   -   X, R⁵ and R¹² are as defined above, and    -   R¹ to R⁴, R⁷ to R¹¹, which may be identical or different,        represent a hydrogen atom, a halogen atom (F, Cl, Br, I), a        hydroxy, alkylenehydroxy, alkynealkylenehydroxy,        alkynehydroxycyclohexyl, alkyl, alkoxy, alkylenealkoxy or        alkylenecyano group, these radicals being straight-chain or        branched, with C1 to C18, the alkylene group being saturated or        unsaturated, the said chain being substituted if necessary with        one or more hydroxy or amino groups; a trifluoromethyl group; a        —COM, —COOM, or —CH₂COOM group (with M representing a hydrogen        atom, a C1 to C18 alkyl group, straight-chain or branched,        substituted if appropriate with one or more hydroxy and/or amino        groups); a nitroso group; a nitro group; or a cyano group;

and the physiologically acceptable salts of these derivatives.

In another preferred family, Y represents, with the adjacent ring, athiazolyl residue and Z═C.

This family corresponds to general formula (III):

in which the substituents have the meanings given above in relation toformula (II).

In yet another family, Y forms, with the adjacent ring, a phenyl groupand Z═N. This family corresponds to formula (IV):

in which the substituents have the meanings given in relation to formula(II).

A group of preferred paullone derivatives in these various familiescorresponds to the case where X represents C═O.

In another group X represents C—S—CH₃ or C—S.

In yet another group X represents —C—NHOH.

In general, the derivatives of the invention advantageously have an IC₅₀with respect to GSK-3β below 10 μM and for many of them below 1 μM, IC₅₀values below 100 nM and even below 10 nM being obtainable.

Particularly preferred paullones of these groups belong to the familiesof formula (II) or formula (III) with R⁹ selected from —NO₂, —CN, —Cl,—Br, —CF₃, C1–C5 alkyl, in particular methyl, or a hydrogen atom, R²and/or R³ selected from alkoxy (of C₁ to C₃ for the alkyl radical), andespecially methoxy, alkylenecyano, vinylalkoxy, or propylene, the othersubstituents being hydrogen.

The invention relates in particular to the use, for manufacturingmedicines that are selectively inhibitors of GSK-3β, of CDK1 and ofCDK5, of paullone derivatives of formula (II) in which X═CO, R⁹ isselected from —NO₂, —CN, —Br, —Cl, —CF₃, H, and R² and/or R³ representH, C₁–C₅ alkoxy, especially methoxy, alkylenecyano, especiallymethylenecyano, the other substituents being hydrogen.

The invention relates quite particularly to the use of paullones offormula (II) in which X═CO,

R⁹ represents —NO₂, —CN, —Br, —Cl or —CF₃, the other substituentsrepresenting hydrogen.

The invention relates again quite particularly to the use of paullonesof formula (II) in which X═CO, R⁹ represents the meanings given aboveand R² and R³ both represent a C₁ to C₅ alkoxy group, in particularmethoxy, or R² represents an alkylenecyano group, in particularethylenecyano.

The invention also relates in particular to the use, for manufacturingmedicines that are selectively inhibitors of GSK-3β, of paullones ofgeneral formula (II) in which X═SCH₃ and R⁹ is as defined above andrepresents in particular a halogen atom, in particular bromine.

According to another embodiment of the invention, paullone derivativesof formula (III) in which R⁹ is as defined above in its preferredmeanings, the other substituents representing a hydrogen atom, are usedfor the manufacture of medicines that are inhibitors of GSK-3β, CDK1 andCDK5, with a greater selectivity with respect to GSK-3β and CDK1.

It is to be noted that 9-cyano-2,3-dimethoxypaullone is a novel productand falls within the scope of the invention on this basis.

The invention therefore makes it possible to produce medicines havingthe selectivity required for a given application.

The medicines manufactured in accordance with the invention using thesaid paullone derivatives are quite particularly suitable for thetreatment of pathologies in which GSK-3β is involved.

This applies in particular to endocrinology, for example in the case ofdiabetes, where GSK-3β inhibitors can be used as insulin-mimetics. Itwill be recalled that insulin acts by a cascade of biochemical eventsleading to inhibition of GSK-3β and that this inhibition is responsiblefor the cells' response to insulin.

These medicines are also of considerable interest for the treatment ofneurodegenerative diseases, such as Alzheimer's disease.Hyperphosphorylation of tau protein caused by CDK5 and GSK-3β can infact be inhibited by paullone derivatives. By administering the drugsmade according to the invention, it is then possible, owing to theirinhibitory effect both on CDK5 and GSK-3β, to preventhyperphosphorylation of tau protein in Alzheimer's patients and combatneurodegeneration and ischaemia.

These drugs are also of considerable interest for treatingmanic-depressive illnesses.

We may also mention their use for the treatment of cancers, where theirinhibitory effect both on GSK-3β and CDK1/2/5, which translates intoapoptosis of the tumour cell, is utilized advantageously.

These drugs also prove effective for the treatment of diseases caused bysingle-celled parasites such as malaria, the trypanosomes, leishmanias,toxoplasmas, pneumocystis etc., or for multicellular parasites, such asfungi and parasitic worms. The genomes of these parasites in factcontain GSK3 homologous genes, but they are different from human GSK-3.

They can also be used in the cardiovascular sphere for treating orpreventing, in particular, atherosclerosis, recurrence of stenosis orangiogenesis, by altering the equilibrium between proliferation andapoptosis, and by controlling the levels of β-catenin.

They will also be used advantageously for the treatment of infectiousdiseases, such as AIDS.

During development of the medicines, the active principles, used intherapeutically effective quantities, are mixed with vehicles that arepharmaceutically acceptable for the chosen route of administration.

Thus, for oral administration, the medicines are prepared in the form ofgelatin capsules, tablets, lozenges, capsules, pills, drops and thelike. Such medicines can contain from 1 to 100 mg of active ingredientper unit.

For administration by injection (intravenous, subcutaneous,intramuscular), the medicines are in the form of sterile or sterilizablesolutions. They may also be supplied in the form of suspensions oremulsions. The doses per dosage unit can vary from 1 to 50 mg of activeingredient. The daily dosage is chosen so as to obtain a finalconcentration of at most 100 μM of paullone derivative in the blood ofthe treated patient.

As a guide, the dosage that can be used in man corresponds to thefollowing doses: thus, in one or more doses of 10 to 50 mg/day, forexample, are administered to the patient for the treatment of tumours orof parasitoses.

In oder to illustrate the invention, without however limiting its scope,other characteristics and advantages are described in the examples givenbelow.

In these examples, reference will be made to FIGS. 1 to 6, showingrespectively:

FIGS. 1A and 1B, inhibitory activity with respect to CDKs, CDK1/CDK2,and GSK-3, of alsterpaullone (FIG. 1A) and of kenpaullone (FIG. 1B) as afunction of the concentration of these paullones,

FIG. 2, the formulae of paullones according to the invention,

FIGS. 3A to 3C, the IC₅₀ values of paullones according to the inventionwith respect to one of the protein kinases GSK3, CDK1/cyclin B, CDK5/p25as a function of their IC₅₀ values with respect to the other 2 proteinkinases,

FIG. 4, the curves showing the inhibition of GSK-3p by alsterpaullonefor competition with ATP,

FIGS. 5A and 5B, the inhibitory effect of alsterpaullone onphosphorylation of tau by GSK-3β in vitro and in vivo, and

FIG. 6, inhibition of the phosphorylation of DARPP-32 by CDK5 on Thr75in vivo by alsterpaullone.

Characterization of the Paullones

Elementary analyses were carried out using a PerkinElmer 2400 instrumentfor elementary analysis of CHN. The ¹H NMR spectra were recorded at 400MHz and the ¹³C NMR spectra at 100 MHz on a Bruker AMX 400 instrument,with tetramethylsilane as internal reference.

The syntheses of the compounds in Table 2 were carried out by themethods described in J. of Medicinal Chemistry mentioned above. Thesynthesis of 2-iodopaullone, 2-bromo-9-nitropaullone,2,3-dimethoxy-9-nitropaullone, 9-cyano-2,3-dimethoxypaullone,7-bromo-5-(4-nitrophenylhydrazono)-4,5-dihydro-1-H-[l]benzazepin-2(3H)-one,7,8-dimethoxy-5-(4-nitrophenylhydrazono)-4,5-dihydro-1H-[1]benzazepin-2(3H)-one,is described in Examples 1 to 6.

Buffers

-   The buffers used have the following compositions:-   Homogenization buffer: 60 mM of β-glycerophosphate, 15 mM of    p-nitrophenylphosphate, 25 mM of Mops (pH 7.2), 15 mM of EGTA, 15 mM    of MgCl₂, 1 mM of DTT, 1 mM sodium vanadate, 1 mM of NaF, 1 mM of    phenylphosphate, 10 μg of leupeptin/ml, 10 μg of aprotinin/ml, 10 μg    of soya trypsin inhibitor/ml and 100 μM of benzamidine.-   Buffer A: 10 mM of MgCl₂, 1 mM of EGTA, 1 mM of DTT, 25 mM of    Tris-HCl pH 7.5, 50 μg of heparin/ml.-   Buffer C: homogenization buffer, but containing 5 mM of EGTA, and    without NaF and protease inhibitors.-   Tris-saline buffer of Tween-20 (TBST): 50 mM of Tris pH 7.4, 150 mM    of NaCl, 0.1% of Tween-20^(r).-   Hypotonic lysis buffer (HLB): 50 mM of Tris-HCl pH 7.4, 120 mM of    NaCl, 10% of glycerol, 1% of Nonidet-P40, 5 nM of DTT, 1 mM of EGTA,    20 mM of NaF, 1 mM of orthovanadate, 5 μM of microcystine, 100 μg/ml    of each of the following products: leupeptin, aprotinin, pepstatin.

Preparations of Kinases and Determinations of the Activities

-   The activities of the kinases were determined in buffer A or C    (unless stated otherwise), at 30° C., at a final ATP concentration    of 15 μM. The values of the blank tests were subtracted and the    activities were calculated in pmol of phosphate incorporated for an    incubation time of 10 minutes. The values of the activities are    generally expressed as a percentage of the maximum activity, i.e. in    the absence of inhibitors.

Control tests were carried out using appropriate dilutions of Me₂SO. Insome cases, as pointed out later, phosphorylation of the substrates isdetermined by autoradiography after SDS-PAGE.

The GSK-3β used is either the enzyme purified from rabbit muscle orexpressed and purified from Sf9 insect cells (Hughes et al., 1992, Eur.J. Biochem., 203: 305, 311). The determinations were carried out with adilution to 1/100 in 1 mg of BSA/ml of DTT 10 mM, with 5 μl of GS-1 40μM as substrate, in buffer A, in the presence of 15 μM [γ³²P] ATP (3000Ci/mol; 1 mCi/ml) in a final volume of 30 μl. After incubation for 30minutes at 30° C., 25 μl aliquots of supernatant were applied to stripsof Whatman P81 phosphocellulose paper, 2.5×3 cm, and 20 seconds laterthe filters were washed 5 times (for at least 5 minutes each time) in asolution of 10 ml of phosphoric acid/liter of water. The wet filtersunderwent counting in the presence of 1 ml of ACS scintillation fluid(Amersham).

The CDK1/cyclin B used was extracted using a homogenization buffer fromstarfish oocytes (Marthasterias glacialis) and purified by affinitychromatography on beads of p9^(CKShsl)-Sepharose, from which the productwas eluted with free p9^(CKShsl), as described by Meijer et al., 1997(Methods in Enzymology, Vol. 283: 113–128), and Borgne et al., 1999, J.Biol. Chem. 274: 11977–11986.

Kinase activity was determined in buffer C, with 1 mg of histone H1/ml,in the presence of 15 μM of [γ³²P] ATP (3000 Ci/mmol; 1 mCi/ml) in afinal volume of 30 μl.

After incubation for 10 minutes at 30° C., 25 μl aliquots of supernatantwere deposited on P81 phosphocellulose papers and treated as describedabove.

The CDK5/p35 was reconstituted by mixing equal quantities of recombinantmammalian CDK5 and of p35 expressed in E. coli in the form of GST(glutathione-S-transferase) fusion protein and purified by affinitychromatography on glutathione-agarose. The enzyme activity of thecomplex was determined in buffer C as described for CDK1/cyclin B.

Phosphorylation of tau in vitro and in vivo

The phosphorylation of tau in vitro was carried out using purifiedGSK-3β and human recombinant tau-32 protein as substrate. Afterincubation for 30 minutes in the presence of various concentrations ofalsterpaullone, in the conditions of investigation of GSK-3β describedabove, reaction of the kinase was stopped by adding Laemmli buffer. Thetau protein was resolved in SDS-PAGE at 10% and its degree ofphosphorylation was visualized by autoradiography.

Cells and viruses: the Sf9 cells (InVitrogen, San Diego, Calif.) werecultivated at 27° C. in a Grace monolayer culture medium (Gibco BRL,Gaithersburg, Md.), supplemented with 10% of fetal calf serum and 50 μgof gentamycin/ml and 2.5 μg of amphotericin/ml. BaculoGold was obtainedfrom PharMingen (San Diego, Calif.), and pVL1392 from InVitrogen.

Transfection of tau: the shortest human tau isoform, with XbaI andBamHI, was excised from a pNG2 bacterial expression vector (Biernat etal., 1993, Neuron, 11: 153–163) and the gene coding for htau23. The genewas inserted in the baculovirus transfer vector pVL1392 cut with thesame endonucleases. The BaculoGold system was used for constructing thevector containing the tau baculovirus. The DNA of BaculoGold is amodified type of baculovirus containing a lethal deletion.

Co-transfection of the DNA of BaculoGold with a complement baculovirustransfer vector makes it possible to recover the lethal deletion fromthis viral DNA and reconstitute viable virus particles carrying thesequence coding for htau23.

The plasmid DNA used for the transfections was purified using QIAGENcartridges (Hilden, Germany).

The Sf9 cells cultivated in monolayers (2×10⁶ cells in a 60 mm cellculture vessel) were co-transfected with baculovirus DNA (0.5 μg ofBaculoGold DNA) and with the pVL1392 derivatives (2 μg) using the methodof calcium phosphate co-precipitation. The infected cells were examinedfor presence of recombinant protein 5 days post-infection by SDS-PAGEand Western blot.

Treatment of Sf9 Cells with Kinase Inhibitors

To determine the effects of inhibitors of aminopurvalanol andalsterpaullone on the phosphorylation of tau, the Sf9 cells infectedwith the baculovirus expressing htau23 were treated 36 hours afterinfection (when the cells have already expressed levels of tausufficient for the development of cellular processes) with 20 μM ofinhibitors for 3 hours before being harvested.

Western Blot of tau:

The Sf9 cells were infected with a recombinant virus at MOI of 1 to 5.

The cell lysates were prepared in the hypotonic lysis buffer (HLB).

After centrifugation for 15 minutes at 16000 g, the supernatant wasrecovered and its NaCl concentration was increased to 500 mM. Thesupernatant was then boiled for 10 minutes and recentrifuged at 16000 gfor 15 minutes. The proteins (3 μg) were resolved by SDS-PAGE,transferred to a PVDF membrane and investigated by Western blot with thefollowing antibodies: AT-8 (1:2000), AT-180 (1:1000), AT-100 (1:500),PHF-1 (1:600) and K9JA anti-tau polyclonal antibody. Immunostaining wasvisualized using an ECL chemiluminescence system (Amersham,Braunschweig, Germany).

Inhibition in Situ of CDK5 in the Striatum

Striatum slices from adult mouse brain are prepared in accordance withthe standard methodology. Monitoring equilibrium in a Krebs bicarbonatebuffer oxygenated with continuous aeration (95% O₂/5% CO₂), the slicesare treated with various concentrations of alsterpaullone, or with 10 μMof roscovitin for 60 minutes, or they are left in the Krebs bicarbonatebuffer for the same length of time. The slices are homogenized bysonication in 1% of SDS at boiling and 50 mM of NaF. The concentrationsof proteins are determined by the BCA method using a standard BSA curve.Equal quantities of proteins (80 μg) were subjected to SDS-PAGE using15% acrylamide gel, transferred by electrophoresis on a nitrocellulosemembrane and subjected to immunoblot with a specific phosphorylationantibody that selectively detects DARPP-32 phosphorylated on Thr75.

EXAMPLE 12-Iodo-7,12-dihydro-indolo[3,2-d][1]benzazepin-(5H)-one(2-iodopaullone)

A mixture of phenylhydrazine (162 mg, 1.5 mmol) and7-iodo-1H-[1]benzazepin-2,5-(3H, 4H)-dione (301 mg, 1 mmol) in glacialacetic acid (10 mL) is stirred for 1 hour at 70° C. Concentratedsulphuric acid (0.1 mL) is added and stirring is continued for 1 hour at70° C. After cooling to room temperature, the mixture is poured into anaqueous solution of sodium acetate at 5%. The precipitate is removed byfiltration, washed with water and crystallized from ethanol. 57%6 ofbeige crystals are obtained, m.p. 303° C. (decomp.); IR (KBr): 3270(NH), 1640 (C═O); ¹H-NMR (DMSO-d₆, 400 MHz): δ (ppm) 3.52 (s, 2H, CH₂),7.04–7.10 (3, 2H), 7.19 (“t”, 7.6 Hz, 1H), 7.43 (d, 8.2 Hz, 1H),7.66–7.69 (m, 2H), 8.07 (d, 1.5 Hz, 1H), 10.17 (s, 1H, lactam NH), 11.66(s, 1H, indole NH); C₁₆H₁₁N₂O (374.18); Calc. C, 51.4, H, 3.0, N, 7.5;Found C, 51.0, H, 3.3, N, 7.2.

EXAMPLE 22-Bromo-7,12-dihydro-9-nitro-indolo[3,2-d][1]benzazepin-6(5H)-one(2-bromo-9-nitropaullone)

7-Bromo-5-(4-nitrophenylhydrazono)-4,5-dihydro-1H-[1]benzazepin-2(3H)-one(389 mg, 1 mmol) is refluxed in diphenyl ether (20 mL) for 2 hours undernitrogen. After cooling to room temperature, hexane (50 mL) is added.The precipitate is filtered, washed with hexane and crystallized fromethanol/toluene. Deep yellow crystals are obtained (35%), m.p. >330° C.;IR (KBr): 3310 (NH), 1670 (C═O); ¹H-NMR (DMSO-d₆, 400 MHz): δ (ppm)=3.69(s, 2H, CH₂), 7.23 (d, 1H, 8.6 Hz), 7.59–7.64 (m, 2H), 7.96 (d, 1H, 2.0Hz), 8.09 (dd, 1H, 9.1/2.0 Hz), 8.77 (d, 1H, 1.5 Hz), 10.32 (s, 1H,lactam NH), 12.46 (s, 1H, indole NH); C₁₆H₁₀BrN₃O₃ (372.19; Calc. C,51.6, H, 2.7, N, 11.2, Br, 21.5; found C, 51.5, H, 3.0, N, 10.8, Br,21.3.

EXAMPLE 32,3-Dimethoxy-7,12-dihydro-9-nitro-indolo[3,2-d][1]benzazepin-6(5H)-one(2,3-dimethoxy-9-nitropaullone)

7,8-Dimethoxy-5-(4-nitrophenylhydrazono)-4,5-dihydro-1H-[1]benzazepin-2(3H)-one(370 mg, 1 mmol) is refluxed in diphenyl ether (20 mL) for 2 hours undernitrogen. After cooling to room temperature, hexane is added (50 mL).The precipitate is filtered, washed with hexane and crystallized fromethanol/toluene. Deep yellow crystals are obtained (63%), m.p. >330° C.;IR (KBr): 3340 (NH), 1660 (C═O); ¹H-NMR (DMSO-d₆, 400 MHz): δ (ppm)=3.58(s, 2H, CH₂), 3.81 (s, 3H, OCH₃), 3.88 (s, 3H, OCH₃), 6.90 (s, 1H), 7.31(s, 1H), 7.31 (s, 1H), 7.59 (d, 1H, 9.2 Hz), 8.05 (dd, 1H, 8.9/2.3 Hz),8.69 (d, 1H, 2.0 Hz), 9.94 (s, 1H, lactam NH), 12.32 (s, 1H, indole NH);C₁₈H₁₅N₃O₅ (353.35); Calc. C, 61.2, H, 4.3, N, 11.9; found C, 60.9, H,4.4, N, 11.8.

EXAMPLE 4 2,3-Dimethoxy-6-oxo-5,6,7,12-tetrahydro-indolo[3,2-d][1]benzazepin-9-carbonitrile(9-cyano-2,3-dimethoxypaullone)

9-Bromo-2,3-dimethoxy-7,12-dihydro-indolo[3,2-d][1]benzazepin-6(5H)-one(387mg, 1 mmol) and copper(I) cyanide (179 mg, 2 mmol) are refluxed for 2hours in N-methyl-2-pyrrolidone (10 mL). After cooling to roomtemperature, water (10 mL) is added and the reaction mixture is stirredfor 15 minutes. The precipitate is removed by filtration and stirred fora further 15 minutes in a mixture of water (10 mL) and ethylenediamine(2.5 mL). The precipitate is eliminated by filtration, washed with a 10%aqueous cyanide solution and crystallized from ethanol/toluene.Colourless crystals (40%) are obtained, m.p. >330° C.; IR (KBr):3300/3200 (NH), 2220 (CN), 1660 (C═O); ¹H-NMR (DMSO-d₆, 400 MHz): δ(ppm)=3.53 (s, 2H, CH₂), 3.80 (s, 3H, OCH₃), 3.87 (s, 3H, OCH₃), 6.89(s, 1H), 7.29 (s, 1H), 7.49 (s, 1H), 7.49 (dd, 1H, 8.6/1.5 Hz), 7.58 (d,1H, 8.2 Hz), 8.27 (s, 1H), 9.89 (s, 1H, lactam NH), 12.10 (s, 1H, indoleNH); C₁₉H₁₅N₃O₃ (333.36); Calc. C, 68.5, H, 4.5, N, 12.6; found C, 68.0,H, 4.6, N, 12.0.

EXAMPLE 57-Bromo-5-(4-nitrophenylhydrazono)-4,5-dihydro-1-H-[1]benzazepin-2(3H)-one

7-Bromo-1H-[1]benzazepin-2,5(3H, 4H)dione (254 mg, 1 mmol) (Kunick C.(1991), Arch. Pharm. (Weinheim) 324, 579–581), 4-nitrophenylhydrazinehydrochloride (284 mg, 1.5 mmol) and sodium acetate (123 mg, 1.5, mmol)are stirred in glacial acetic acid (10 ml) for 1 hour at 70° C.

After cooling to room temperature, the mixture is poured into a 5%aqueous solution of sodium acetate (20 ml). The precipitate is recoveredby filtration, washed with water, and crystallized from ethanol. Yellowcrystals are obtained, with a yield of 52%.; m.p. 300° C. (decomp.); IR(KBr)/3220 (NH), 1670 (C═O); ¹H-NMR (DMSO-d₆, 400 MHz):

−δ (ppm)=2.56–2.59 and 3.02–3.06 (m, AA′XX′, 4H, CH₂—CH₂), 6.99 (d, 1H,8.1 Hz), 7.33 (d, 2H, 9.2 Hz), 7.56 (dd, 1H, 8.7/2.6 Hz), 7.75 (d, 1H,2.0 Hz), 8.16 (d, 2H, 9.6 Hz), 7.56 (dd, 1H, 8.7/2.6 Hz), 7.75 (d, 1H,2.0 Hz), 8.16 (d, 2H, 9.6 Hz), 9.87 (s, 1H, NH), 10.19 (s, 1H, NH);C₁₆H₁₅BrN₄O₃ (389.22); Calc. C, 49.4, H, 3.4, N, 14.4, Br, 20.5; FoundC, 49.1, H, 3.4, N, 14.1, Br, 20.2.

EXAMPLE 67,8-Dimethoxy-5-(4-nitrophenylhydrazono)-4,5-dihydro-1H-[1]benzazepin-2(3H)-one

7,8-Dimethoxy-1H-[1]benzazapin-2,5[3H, 4H)dione (235 mg, 1 mmol)(Schultz C. et al., (1999) J. Med. Chem., 42, 2909–2919),4-nitrophenylhydrazine hydrochloride (569 mg, 3 mmol) and sodium acetate(246 mg, 3 mmol) are stirred in glacial acetic acid (10 ml) for 1 hourat 70° C.

After cooling to room temperature, the mixture is poured into a 5%aqueous solution of sodium acetate (20 ml). The precipitate is recoveredby filtration, washed with water, and crystallized from ethanol. Yellowcrystals are obtained, with a yield of 60%; m.p. 286° C. (decomp.); IR(KBr)/3260/3180 (NH), 1680 (C═O); ¹H-NMR (DMSO-d₆, 400 MHz):

−δ (ppm)=2.53–2.56 and 2.99–3.03 (m, AA′XX′, 4H, CH₂—CH₂), 3.77 (s, 3H,OCH₃), 3.81 (s, 3H, OCH₃), 6.65 (s, 1H), 7.20 (s, 1H), 7.32 (d, 2H, 9.2Hz), 8.13 (d, 2H, 9.2 Hz), 9.53 (s, 1H, NH), 10.06 (s, 1H, NH);C₃₈H₁₈N₄O₅ (370.38); calc. C, 58.4, H, 4.9, N, 15.1; found C, 57.8, H,4.9, N, 14.8.

EXAMPLE 7 Investigation of the Inhibition of GSK-3β, of CDK5/p35 and ofCDK1/Cyclin B by the Paullones

Alsterpaullone

The activity of alsterpaullone was studied on several highly purifiedkinases.

The kinase activities were determined with a suitable substrate (GSK-3β:GS1 peptide; CDKs: histone Hi) in the presence of 15 μM of ATP and atincreasing concentrations of alsterpaullone and kenpaullone.

The activity is expressed as a percentage of the maximum activity.

The results obtained are shown in FIG. 1 and Table 1. The IC₅₀ valueswere calculated from the dose/response curves and are expressed in μM.

TABLE 1 Alsterpaullone Enzyme IC50 (μM) CDK 1/cyclin B

CDK 2/cyclin A

CDK 2/cyclin E

CDK 4/cyclin D1

CDK 5/p35

GSK-3 α

GSK-3 β

erk 1

erk 2

c-raf

MAPKK

c-jun N-terminal kinase

protein kinase C α

protein kinase C β1

protein kinase C β2

protein kinase C γ

protein kinase C δ

protein kinase C ε

protein kinase C η

protein kinase C ζ

CAMP-dependent protein kinase

cGMP-dependent protein kinase

casein kinase 1

casein kinase 2

Insulin receptor tyrosine kinase

It can be seen that most of the kinases tested are slightly inhibited ornot at all (IC₅₀>10 μM).

It will be noted that as well as the effect on CDK1/cyclin B,alsterpaullone inhibits CDK2/cyclin A, CDK2/cyclin E, CDK5/p25 andGSK-3α/GSK-3β (IC₅₀ respectively 15, 200, 40 and 4 nM).

Other Paullones According to Formula I

Tables 2A and 2B given below and FIG. 2 show the inhibitory effect ofpaullones used according to the invention with respect to GSK-3β,CDK5/p25 and CDK1/cyclin B.

TABLE 2A Number Compounds GSK3 CDK1 CDK5 98 N 215 alsterpaullone(9-Nitropaullone) 0.004^(d) 0.035^(b) 0.040^(b) 98 N 210 9-cyanopaullone0.010^(b) 0.024^(b) 0.044^(b) 98 N 356 2,3-dimethoxy-9-nitropaullone0.013^(b) 0.024^(b) 0.021^(b) 98 N 217 9-cyano-2,3-dimethoxypaullone0.018^(b) 0.044^(b) 0.060^(b) 96 N 619 kenpaullone (9-Bromopaullone)0.023^(b) 0.400^(c) 0.350^(c) 97 N 343 9-chloropaullone 0.024^(b)0.600^(c) 0.300^(c) 97 N 487 9-trifluoromethylpaullone 0.030^(b)0.400^(c) 0.500^(c) 98 N 3573-(6-oxo-9-trifluoromethyl-5,6,7,12-tetrahydro- 0.033^(b) 0.047^(b)0.033^(b) indolo[2-3-d] [1]benzazepin-2-yl)-propionitrile (B) 98 N 0022,3-dimethoxy-9-trifluoromethylpaullone 0.075^(b) 0.280^(c) 0.430^(c) 98N 048 9-bromo-12-methyloxycarbonylmethylpaullone 0.075^(b) 1.400^(d)350.000^(c) 97 N 353 9-fluoropaullone 0.080^(b) 1.600^(d) 1.300^(d) 97 N608 9-bromo-2,3-dimethoxypaullone 0.100^(c) 0.200^(c) 0.500^(c) 97 N 4839-bromo-2,3-dimethoxypaullone 0.120^(c) 3.000^(d) 8.000^(d) 97 N 3529-methylpaullone 0.130^(c) 2.000^(d) 6.300^(d) 97 N 345 10-bromopaullone0.140^(c) 1.300^(d) 2.700^(d) 97 N 318 2-bromopaullone 0.200^(c)3.300^(d) 5.000^(d) 97 N 344 11-chloropaullone 0.200^(c) 1.400^(d)2.900^(d) 98 N 351 2-(3-hydroxy-l-propinyl)-9-trifluoromethylpaullone(C) 0.200^(c) 0.300^(c) 2.000^(d) 98 N 354 2-bromo-9-nitropaullone0.200^(c) 0.053^(b) 0.120^(c) 98 N 259 2-iodopaullone 0.250^(c)3.700^(d) 7.400^(d) 98 N 051 9-bromo-12-(2-hydroxyethyl)-paullone0.300^(c) 3.000^(d) 140.000^(c) 98 N 211 9-bromo-12-methylpaullone0.400^(c) 6.200^(d) 400.000^(c) 98 N 350(E)-3-(6-oxo-9-trifluoromethyl-5,6,7,12-tetrahydro- 0.400^(c) 0.270^(c)9.500^(d) indolo[2-3-d] [1]benzazepin-2-yl)-acrylonitrile (E) 97 N 4869-bromo-5-(methyloxycarbonylmethyl)paullone 0.500^(c) 6.400^(d)5.300^(d) 98 N 223 11-methylpaullone 0.500^(c) 3.000^(d) 9.000^(d) 97 N317 paullone (A) 0.620^(c) 7.000^(d) 10.100^(c) 98 N 22511-ethylpaullone 0.700^(c) 3.800^(d) 23.000^(c) 98 N 2169-bromo-7,12-dihydro-6-(hydroxyamino)-indolo[2-3-d] [1] benzazepine (F)0.750^(c) 1.000^(d) 2.100^(d) 97 N 609 2,9-dibromopaullone 0.800^(c)0.300^(c) 10.000^(c) 97 N 347 11-bromopaullone 0.900^(c) 1.300^(d)1.400^(d) 97 N 485 2,3-dimethoxypaullone 0.900^(c) 4.300^(d) 5.400^(d)98 N 262 (E)-3-(6-oxo-9-trifluoromethyl-5,6,7,12-tetrahydro- 0.900^(c)4.300^(d) 130.000^(c) indolo[2-3-d] [1]benzazepin-2-yl)-acrylic acidmethyl ester (D) 97 N 6109-bromo-7,12-dihydro-6-methylthio-indolo[2-3-d] [1]benzazepine (G)1.200^(d) 43.000^(c) 450.000^(c) 98 N 358(E)-2-(3-oxo-1-butenyl)-9-trifluoromethylpaullone (H) 1.400^(d)0.320^(c) 34.000^(c) 98 N 213 9-bromo-12-ethylpaullone 1.500^(d)23.000^(c) 260.000^(c) 97 N 612 9-bromo-7,12-dihydro-indolo[2-3-d][1]benzazepine-6 (5H)-thione (I) 2.000^(d) 2.300^(d) 8.000^(d) 98 N 0472-bromo-9-trifluoromethylpaullone 2.000^(d) 0.240^(c) 3.000^(d) 98 N 3492-[2-(1-hydroxycyclohexyl)-ethinyl]-9-trifluoromethyl-paullone (J)2.000^(d) 3.200^(d) 8.300^(d) 97 N 613 9-bromo-5-methylpaullone2.100^(d) 20.000^(c) 130.000^(c) 97 N 351 9-methoxypaullone 2.200^(d)0.900^(c) 2.100^(d) 98 N 236 2-iodo-9-trifluoromethylpaullone 2.200^(d)0.700^(c) 7.000^(d) 98 N 0469-bromo-12-(tert.-butyloxycarbonyl)-paullone 2.300^(d) 70.000^(c)800.000^(c) 98 N 212 9-bromo-12-(2-propenyl)-paullone 4.000^(d)60.000^(c) 240.000^(c) 97 N 482 9-bromo-4-hydroxypaullone 4.300^(d)40.000^(c) 850.000^(c) 98 N 049 8,10-dichloropaullone 5.000^(d)2.500^(d) 350.000^(c) 98 N 001 5-benzyl-9-bromopaullone 10.000^(c)35.000^(c) 270.000^(c) 97 N 607 9-bromo-4-methoxypaullone 16.000^(c)250.000^(c) 400.000^(c) 98 N 224 9-bromo-5-ethylpaullone 24.000^(c)470.000^(c) 300.000^(c) 98 N 2099-bromo-5,7-bis-(tert.-butyloxycarbonyl)-paullone 130.000^(c) 80.000^(c)>1000^(c) 97 N 484 4-methoxypaullone 140.000^(c) 430.000^(c) 100.000^(c)98 N 347 9-bromo-5,6,7,12-tetrahydro-benzo[6-7]cyclohept 180.000^(c)51.000^(c) 860.000^(c) [1,2.b] indole (K) 98 N 2142-phenyl-4-(2-thienyl)-5H-pyrido[2-3-d] [1] 350.000^(c) 33.000^(c)700.000^(c) benzazepine-6(7H)-thione (L) 98 N 2089-bromo-5,7,12-tri-(tert.-butyloxycarbonyl)-paullone 500.000^(c)150.000^(c) 1000.000^(c) 97 N 6119-bromo-5,12-bis-(tert.-butyloxycarbonyl)-paullone 640.000^(c)1000.000^(c) >1000^(c) 98 N 220 4-(4-chlorophenyl)-2-(2-naphthyl)->1000^(c) >1000^(c) >1000^(c) 5H-pyrido[2-3-d][1]benzazepine-6(7H)-thione (M) 98 N 3485,6,7,12-tetrahydro-benzo[6-7]cyclohept[1,2-b]indole (N) >1000^(c)130.000^(c) >1000^(c) ^(a)<0.01 μM: ^(b)0.01–0.1 μM: ^(c)0.1–1 μM^(d)1–10 μM ^(e)10 μM

Table 2B also shows the IC₅₀ values for GSK3, CDK1 and CDK5 for otherpaullone derivatives used according to the invention. The meaningsrepresented by the substituents are shown, for each compound, when it isnot a hydrogen atom.

TABLE 2B Compounds GSK3 CDK1 CDK5 X = —SCH3; R⁹ = —Br 0.034 43.000160.000 Y = thienyl residue; R⁹ = —Br 0.120 0.600 4.000 R² =—CH═CH—CO—CH₃; R⁹ = —CF₃ 0.350 4.300 15.000 R⁹ = R¹¹ = —F 0.400 3.40010.000 Y = thienyl; R⁹ = —Cl 0.400 0.500 5.000 Y = thienyl; R⁹ = —CH₃1.300 4.000 40.000 R² = R³ = —OH 2.200 32.000 42.000 R² = —I; R⁹ = —Br4.200 0.320 30.000 Z = pyridyl 5.500 2.200 3.300

For comparing the effects of the active compounds on GSK-3β and theCDKs, FIGS. 3A to 3C show the IC₅₀ values with respect to each enzyme(CDK1/cyclin B, CDK5/p25 and GSK-3β) as a function of the IC₅₀ valueswith respect to two other kinases.

This analysis shows that the efficacies of the paullones with respect toCDK1 and CDK5 are closely related, but are less so with respect toGSK-3p and the CDKs.

EXAMPLE 8 Study of the Inhibition of GSK-3β by the Paullones inCompetition with ATP

FIG. 4 presents kinetic data from determination of the activity ofGSK-3β at different concentrations of alsterpaullone. The enzymeactivities are determined as described above. FIG. 4A: primary curve 1/Vversus 1/ATP. The ATP concentrations in this reaction mixture vary from0.5 to 4 μM. The concentration of GS-1 is kept constant at 4 μM. Theinset shows the gradients as a function of the concentration from theprimary curves. The apparent inhibition constant (Ki) is shown by anarrow.

The kinetic experiments demonstrate that alsterpaullone also competeswith ATP for binding to GSK-3β.

The apparent inhibition constant (ki) is 20 nM.

EXAMPLE 9 Study of the Inhibition, by Alsterpaullone, of thePhosphorylation of tau in vitro and in vivo by GSK-3β

The inhibitory effect of alsterpaullone on the activity of GSK-3β wasdetermined on a microtubule-binding tau protein. The human recombinanttau protein expressed in bacteria can be phosphorylated in vitro byGSK-3β and this phosphorylation is inhibited in a dose-dependent mannerby alsterpaullone with an IC₅₀ close to 33 nM.

FIG. 5A shows the results of resolution by SDS-PAGE, followed byautoradiography. FIG. 5B gives the results obtained with Sf9 cellsexpressing htau23 (untreated control), either exposed to alsterpaulloneor to aminopurvalanol for 3 hours. The cell lysates (3 μg htau23) areresolved by SDS-PAGE, stained with Coomassie blue or subjected to animmunoblot with different antibodies: K9JA (pantau antibody) whichrecognizes tau independently of phosphorylation, AT100, which recognizestau phosphorylated on Thr212 and Ser214, this reaction being veryspecific to the tau protein of Alzheimer's, PHF-1 (phosphorylated onSer396/Ser-404, AT8 (phosphorylated on Ser202/Thr205), and AT180(phosphorylated on Thr231/Ser235).

EXAMPLE 10 Investigation of Inhibition of the Phosphorylation ofDARPP-32 by CDKs in vivo

The role of the protein DARPP-32, as a physiological substrate ofCDKs/p25, was recently identified by Bibble J. A. et al. (1999), Nature,402, 669–671. This protein becomes an inhibitor of PKA when it isphosphorylated on Thr75 by CDK5/p25. In vivo, phosphorylation is notobserved on this site in p35⁻/⁻ tissues. To determine the CDK5inhibiting capacity of alsterpaullone in the brain, slices of striatum(region of the brain expressing DARPP-32) were treated with differentconcentrations of alsterpaullone. The slices were incubated with 0.1, 10and 50 μM of alsterpaullone, for 60 minutes. The degree ofphosphorylation of DARPP-32 on Thr75 was monitored by Western blot witha phospho-specific antibody and evaluated by quantification of thebands. The results obtained are shown in FIGS. 5A and 5B respectively.It can be seen that alsterpaullone is capable of inhibiting thephosphorylation of DARPP-32 in situ.

EXAMPLE 11 Preparation of a Capsule Using 9-Nitropaullone as ActiveIngredient

20 mg of 9-nitropaullone is mixed with the standard excipients used forthe manufacture of gelatin capsules.

What is claimed is:
 1. A method of treatment of diabetes, comprisingadministering to a patient in need thereof i. an effective amount of amedicine comprising a paullone derivative corresponding to generalformula (I):

in which X represents a C═O, C—S—CH₃, C—S, or —C—NHOH group; Zrepresents C or N; Y represents, with the adjacent ring, a phenyl orthiazolyl residue; the ring or rings constituting these derivativesbeing optionally substituted by one or more: halogen atoms, hydroxy,alkylenehydroxy, alkynealkylenehydroxy, alkynehydroxycyclohexyl, alkyl,alkoxy, alkylenealkoxy, alkylenecyano groups, the alkylene group beingsaturated or unsaturated, these radicals being straight-chain orbranched, of C1 to C18, the said chain being optionally substituted withone or more hydroxy or amino groups; one or more trifluorometbyl; —COM;—COOM; or —CH₂COOM groups (with M representing a hydrogen atom a C1 toC18 alkyl group, straight-chain or branched, optionally substituted withone or more hydroxy, amino; nitroso; nitro; or cyano groups; R⁵represents a hydrogen atom or a C₁ to C₅ alkyl group, R¹² represents ahydrogen atom, or a —C—CO₂—(CH₃)₃ group, and the physiologicallyacceptable salts of these derivatives, and ii. a pharmaceuticallyacceptable carrier or vehicle.
 2. The method according to claim 1,characterized in that the paullone derivatives correspond to formula(II)

in which, X represents a C═O, C—S—CH₃, C—S, or —C—NHOH group, R⁵represents a hydrogen atom or a C₁ to C₅ alkyl group and R¹² representsa hydrogen atom, or a —C—CO₂—(CH₃)₃ group, and R¹— to R⁴, R⁷ to R¹¹,which may be identical or different, represent a hydrogen atom, ahalogen atom (F, Cl, Br, I), a hydroxy, alkylenehydroxy,alkynealkylenehydroxy, alkynehydroxycyclohexyl, alkyl, alkoxy,alkylenealkoxy or alkylenecyano group, these radicals beingstraight-chain or branched, with C1 to C18, the alkylene group beingsaturated or unsaturated, the said chain being optionally substitutedwith one or more hydroxy or amino groups; a trifluorometbyl group; a—COM, —COOM, or —CH₂COOM group (with M representing a hydrogen atom, aC1 to C18 alkyl group, straight-chain or branched, optionallysubstituted with one or more hydroxy or amino groups); a nitroso group;a nitro group; or a cyano group; and the physiologically acceptablesalts of these derivatives.
 3. The method according to claim 1,characterized in tat the paullone derivatives correspond to formula(III),

in which X represents a C═O, C—S—CH₃, C—S, or —C—NHOH group, R⁵represents a hydrogen atom or a C1 to C5 alkyl group and R¹² representsa hydrogen atom, or a —C—CO₂—(CH₃)₃ group, and R², R³, R⁷ to R¹¹, whichmay be identical or different, represent a hydrogen atom, a halogen atom(F, Cl, Br, I), a hydroxy, alkylenehydroxy, alkynealkylenehydroxy,alkynehydroxycyclohexyl, alkyl, alkoxy, alkylenealkoxy or alkylenecyanogroup, these radicals being straight-chain or branched, with C1 to C18,the alkylene group being saturated or unsaturated, the said chain beingoptionally substituted with one or more hydroxy or amino groups; atrifluorometbyl group; a —COM, —COOM, or —CH₂COOM group (with Mrepresenting a hydrogen atom, a C1 to C18 alkyl group, straight-chain orbranched, optionally substituted with one or more hydroxy or aminogroups); a nitroso group; a nitro group; or a cyano group.
 4. The methodaccording to claim 1, characterized in that The paullone derivativescorrespond to general formula (IV):

in which X represents a C═O, C—S—CH₃, C—S, or —C—NHOH group, R⁵represents a hydrogen atom or a C₁ to C₅ alkyl group and R¹² representsa hydrogen atom, or a —C—CO₂(CH₃)₃ group, and R¹ to R⁴, R⁷ to R¹¹, whichmay be identical or different, represent a hydrogen atom, a halogen atom(F, Cl, Br, I), a hydroxy, alkylenehydroxy, alkynealkylenehydroxy,alkynehydroxycyclohexyl, alkyl, alkoxy, alkylenealkoxy or alkylenecyanogroup, these radicals being straight-chain or branched, with C1 to C18,the alkylene group being saturated or unsaturated, the said chain beingoptionally substituted with one or more hydroxy or amino groups; atrifluorometbyl group; a —COM, —COOM, or —CH₂COOM group (with Mrepresenting a hydrogen atom, a C1 to R18 alkyl group, straight-chain orbranched, optionally substituted with one or more hydroxy or aminogroups); a nitroso group; a nitro group; or a cyano group.
 5. The methodaccording to claim 2, wherein X represents C═O.
 6. The method accordingto claim 2, wherein X represents C—S—CH₃, or C—S.
 7. The methodaccording to claim 2, wherein X represents —C—NHOH.
 8. The methodaccording to claim 2, wherein, R⁹ is selected from —NO₂, —CN, —Cl, —Br,—CF₃, C1 to C5 alkyl or a hydrogen atom, R² and R³ are selected fromC₁–C₃ alkoxy, alkylenecyano, vinylalkoxy, or propylene.
 9. The methodaccording to claim 8, wherein X═CO, R⁹ is selected from —NO₂, —CN, —Br,—Cl, —C₃, H, and R² and R³ are independently selected from H, C₁–C₃alkoxy, alkylenecyano.
 10. The method according to claim 9,characterized in that X=CO, R⁹ represents —NO₂, —CN, —Br, —Cl or —CF₃.11. The method according to claim 9, characterized in that X=CO, R⁹represents —NO₂, —CN, —Br, —Cl or —CF₃, and R² and R³ both represent aC₁ to C₃ alkoxy group, or R² represents an alkylenecyano group.
 12. Themethod according to claim 6, characterized in that R⁹ is selected from—NO₂, —CN, —Br, —Cl, —CF₃, H.
 13. A method treatment of a diseaseselected from diabetes and manic-depressive illnesses, comprisingadministering to a patient in need thereof -nitro-7,12-dihydroindolo[3,2-d] [1]-benzazepin-6(5H)-one.
 14. The method according to claim 2,in which X═SCH₃ and R⁹ is selected from —NO₂, —CN, —Br, —Cl, —CF₃, H.15. The method according to claim 3, in which R₉ is selected from —NO₂,—CN, —Br, —Cl, —CF₃, H, atom.
 16. The method according to claim 1,characterized in that the medicines are prepared for oraladministration, in the form of gelatin, tablets, lozenges or capsules.17. The method according to claim 1, characterized in that the drugs areprepared for administration by injection, in the form of solution.